1. Field of the Invention
The present invention relates to methods for culturing stem cells in modeled microgravity conditions, including embryonic, adult, multipotent hematopoietic progenitor cells and cancer stem cells. The present invention also relates to methods for proliferating stem cells by culturing stem cells, including embryonic, adult, multipotent hematopoietic progenitor cells and cancer stem cells, under conditions of microgravity. The current invention also relates to methods for increasing telomerase activity and telomere length. Furthermore, the current invention relates to methods for culturing cancer stem cells under microgravity conditions thereby increasing their susceptibility to chemotherapeutic agents. The present invention also relates to methods for mass producing cellular factors by culturing stem cells under conditions of microgravity. Additionally, the current invention relates to a method for improving the treatment outcome of cancer in a mammal by subjecting the mammal to simulated microgravity followed by administering a chemotherapeutic agent to the mammal. The current invention also relates to a method for testing effectiveness of chemotherapy drugs on cancer stem cells.
2. Background Art
Stem cells show potential for many different areas of health and medical research. Some of the most serious medical conditions, such as cancer and birth defects, are caused by problems that occur somewhere in the process of stem cell differentiation or maintenance. Broadly, there are two different types of stem cells, embryonic stem cells and adult stem cells. Embryonic stem cells are found in blastocysts and have the ability to differentiate into all of the specialized embryonic tissues. Adult stem cells are undifferentiated cells found throughout the body after embryonic development. Adult stem cells are able to divide and replenish dying cells and regenerate damaged tissue. Furthermore, adult stem cells can maintain the normal turnover of regenerative organs such as blood, skin and intestinal tissue. Adult stem cells have the ability to divide and self-renew indefinitely and are able to generate all of the cell types of the organ from which they originate.
Stem cells can be classified as being totipotent, pluripotent, multipotent or unipotent based on their potential to differentiate into different cell types. Totipotent stem cells are produced from the fusion of gametes and the first few divisions of the fertilized egg. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells can differentiate into cells from any of the three germ layers. Multipotent calls can produce only cells of a closely related family. Unipotent cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells. Most adult stem cells are lineage restricted multipotent stem cells, and are referred to by their tissue of origin. Pluripotent adult stem cells are rare and generally small in number but can be found in a number of tissues including umbilical cord blood (Ratajczak M. Z., et al., Leukemia 21(5): 860-867 (2007)). There are several different types of adult stem cells including, but not limited to, adipose derived stem cells (Zuk, P. A., et al., Tissue Engineering 7:211-216) (2001)), epithelial stem cells, hematopoietic stem cells, mammary stem cells (Shackleton, M., et al., Breast Cancer R E. 7:86-95 (2005)), mesenchymal stem cells, endothelial stem cells, neural stem cells (Alvarez-Bullta, A., et al., Brain Res. Bull. 57:751-758 (2002)), olfactory stem cells (Murrel, W., et al., Dev. Dyn. 233:496-515 (2005)), testicular stem cells, dental pulp derived stem cells, and umbilical cord blood hematopoietic progenitor cells.
When an adult stem cell divides, it creates another cell like itself and a cell more differentiated than itself. This process of asymmetric cell division, gives rise to one identical daughter cell and one early transient-amplifying cell (early TA), which possesses high proliferative capacity. Through a series of cell divisions, the early TA cell gives rise to a late TA cell followed by a tissue-specific progenitor cell and finally to the bulk of differentiated cells that make up the organ or tissue (Ribacka, C., et al. Ann. Med. epub ahead of print: 1-10 (2008)).
Many approaches have been employed in the in vitro expansion of stem cells in the last decade, and they can be generally divided into two categories. The first category is treatment of stem cells with various combinations of stem cell expansion factors. Treatment with combinations of stem cell expansion factors has been shown to increase a progenitor/stem cell population by 2- to 30-fold in the relatively short period of 10 to 14 days. However, it is difficult to maintain stem cell activity in long-term cultures even if the total number of cells could be expanded. The second category involves using stromal cells. Several methods of in vitro expansion using primary stromal cells have been reported (Gan et al., Blood 90:641 (1997); Yamaguchi et al., Exp. Hematol. 29:174 (2001)). When stem cells were co-cultured with primary stromal cells, the stem cells were expanded for 2 to 4 weeks. It is hypothesized that stromal cells provide cell-cell or cell-extracellular matrix cues that may help expand and maintain the stem cells in an undifferentiated state. However, stem cells have frequently lost their pluripotency during in vitro expansion. Maintenance of pluripotency, number, and proliferative activity of stem cells is critical for transplantation. In addition, removal of stromal cells prior to transplantation also remains a caveat of this category.
Another type of adult stem cell is the cancer stem cell. It is known that a small percentage of cells within an established tumor have the properties of stem cells. These solid tumor stem cells give rise to both more tumor stem cells and to the majority of cells in the tumor that have lost the capacity for extensive proliferation and the ability to give rise to more tumors. Thus, solid tumor heterogeneity reflects the presence of tumor cell progeny arising from a solid tumor stem cell. Presently used means of cancer treatment would thus leave the cancer stem cells unharmed and allow them to induce regrowth of the tumor after seemingly effective treatment. Radiation therapy and most current chemotherapeutic agents target replicating cells, with adult stem cells demonstrating remarkable resistance to these treatments (Ribacka, C., et al. Ann. Med. epub ahead of print: 1-10 (2008)).
Thus, there is a need to establish stem cell cultures that can both expand stem cells and maintain stem cells in their undifferentiated state. In addition, there is a need for methods to increase the sensitivity of cancer stem cells to chemotherapy.